pCambia Vectors

Information on our latest vectors, which contain GUSPlus™, may be found in
the
BioForge.
You can request these vectors separately (they are available under the open
source terms of a BiOS license (info) ) or simultaneously when
registering for the TransBacter Project , in which
we're improving the alternative to Agrobacterium transformation for
more complete freedom to operate and freedom to cooperate.

Much of the information below relates to older pCAMBIA vectors, which will
soon be available under a new MTA with similar conditions to facilitate
cooperative development.

Plant transformation is now routine in hundreds of laboratories worldwide,
using bacterially-mediated or direct DNA transfer methods such as bombardment.
These methods have both technical and intellectual property limitations (see the
BioForge TransBacter Project , in which we're improving
the alternative to Agrobacterium transformation for more complete
freedom to operate). One of the biggest technical limitations laboratories face
is that many vectors still used are historical relics with substandard features
that make DNA constructions awkward or cumbersome:

low-copy origin of replication resulting in low yield DNA preps;

unstable replicons, causing variable loss of the plasmid during propagation;

large size;

lack of convenient restriction sites for manipulation;

limited choice of selectable markers for both bacteria and plants;

lack of simple ways to construct reporter gene fusions;

The pCAMBIA vector backbone is derived from the pPZP vectors (constructed by
Hajdukiewicz, Svab & Maliga, see
References). While not perfect and having
technical and IP limitations (see the BioForge GUSPlus
Project , in which we're improving reporter genes and selection methods for
more complete freedom to operate), pCAMBIA vectors offer:

high copy number in E.coli for high DNA yields

pVS1 replicon for high stability in Agrobacterium

small size, 7-12kb depending on which plasmid

restriction sites designed for modular plasmid modifications and small but
adequate poly-linkers for introducing your DNA of interest

bacterial selection with chloramphenicol or kanamycin

plant selection with hygromycin B or kanamycin (phosphinothricin selection
was discontinued at the request of the IP owner, Bayer, after the initial
distribution in 1997)

A few points about the pCAMBIA cloning strategy:

The pUC18 polylinker was used in some vectors, but pUC8 and pUC9 polylinkers
were also used to simplify the choice of cloning enzyme. In the age of PCR, it
is no longer necessary to have a large number of cloning sites. The smaller
polylinkers also eliminate potential conflicts from sites such as SphI
(which has an ATG) or XbaI (which has a TAG). This makes other sites in
the vector more useful (such as the SphI site outside the right T-DNA
Border, or the SacII site outside the left T-DNA Border).

Plant selection genes in the pCAMBIA vectors are driven by a double-enhancer
version of the CaMV35S promoter and terminated by the CaMV35S polyA signal.
NOTE that this 35S promoter can have an enhancer effect on the
expression of other genes in the same cassette, so gene expression results using
pCAMBIA derivatives in which portions of this promoter are still present should
be interpreted with caution. Furthermore, it is your responsibility to check
whether the 35S promoter or any other components you use are subject to patents
in your country. You can find help with this at
CAMBIA's Patent Lens website.

Reporter genes feature a hexa-Histidine tag at the C-terminus to enable
simple purification on immobilised metal affinity chromatography resins. The
sequence for this tag occurs between the first NheI site (there is a
second NheI site in the pVS1-rep that we didn't eliminate) and the
unique PmlI site. Genes of interest may be inserted in place of the
reporter gene. Insertion without a stop codon and in frame at the (first)
NheI site will append a hexa-Histidine tag to your protein of interest.
Insertion without a stop codon and in frame at the PmlI site will
append a stop codon. Insertion at the BstEII site will add neither a
tag nor a stop codon (so you may want to ensure that a sequence inserted here
contains a stop codon).

Nomenclature of pCAMBIA vectors:

The four digit numbering system works as follows:

First digit - indicates plant selection: 0 for absence; 1
for hygromycin resistance; 2 for kanamycin; and 3 for phosphinothricin (the
vectors containing the phosphinothricin resistance gene are no longer available
from CAMBIA at the request of Bayer, which owns patents restricting its use in
some countries).

Fifth digit - notes some other special feature. So far this
has been used only with: pCAMBIA1305.1 and plasmids derived from it, where the
.1 denotes the absence of a signal peptide from the GUSPlus™ protein; and
pCAMBIA1305.2 where the .2 denotes the presence of the GRP signal peptide for
in planta secretion of the GUSPlus™ protein.

Lagging letter - X indicates that the reporter gene lacks
its own start codon and the vector is for creating fusions to the reporter; Z
indicates presence of a functional lacZa for blue-white screening;
a/b/c indicates the reading frame for fusions with the Fuse and Use vectors.

Important note: Due to resource limitations, not all
possible vector feature combinations have been created at CAMBIA. You may
initially be disappointed to find that we don't have, for example, a
pCAMBIA2205.2. The vectors were designed however, such that it should be a
relatively simple matter for a researcher needing such a vector to construct it
from the components in other vectors. If you have created a pCAMBIA
vector derivative that other researchers will find useful and you want
to share with other researchers, email us.

This vector is a part of a new series of pCAMBIA GT-BACK vectors (gene
Transfer-Bacterial Acquired Competence with kanamycin selection) that
encompasses a modified version of pCAMBIA 1105.1 and a fragment of the Ti
plasmid (derived from pTiBo542) containing only the virA, virB, virC, virD,
virE, virG, virK & virJ operons. This new unitary vector
allows the DNA transfer capability to be moved into, and stabilized in, a much
wider range of bacteria and it will be provided as an open source toolkit.
The added-value features of the new vector over the Transbacter Technology
are: • It can be distributed as spots of dilute DNA on paper
(e.g. on a letter) eliminating the need for compliance with importation
controls on living organisms, and other quarantine issues. This is the means by
which literally thousands of laboratories worldwide have obtained (and further
sent out) pCAMBIA vector sets. • GT-BacK vector lacks the RK2 derived
oriT that Transbacter carried, thus eliminating or reducing the ability
of the plasmid to be conjugated and transmissible to other hosts by RK
conjugation functions. • It has a broad host range replication origin
from pVS1 allowing much wider spectrum of bacteria to be explored as gene
transfer vectors, allowing choices of benign symbionts which do not impose
physical or genetic stresses on plants.

If you wish to discuss or query any CAMBIA materials please
login to
the

pCAMBIA0380; pCAMBIA0390

These vectors contain a range of restriction sites on either side of the pUC8
(0380) or pUC9 (0390) polylinker, making them suitable for advanced construction
purposes with users inserting their own promoters, selection genes, reporter
genes, etc. The only functional signals between the T-DNA borders are the start
and stop codons, the histidine tag, and the NOS-poly(A) signal. All the standard
features of the pCAMBIA backbone are present: kanamycin bacterial selection,
high copy number in E. coli, and stable replication in A.
tumefaciens.

These vectors contain minimal heterologous sequences for plant transformation
and selection of transformants; they allow insertion of desired genes for
transformation into plants but require all promoter and terminator sequences for
plant expression of newly cloned genes.

The minimal selection vectors have one of two plant selection genes:
hptII encoding resistance to hygromycin, or nptII encoding
resistance to kanamycin. In both cases the selection gene is driven by a
double-enhancer version of the CaMV35S promoter. These genes have been subjected
to site-directed mutagenesis to eliminate interfering restriction sites within
the coding sequence by silent changes. Two different bacterial resistance
markers are provided (kanamycin or chloramphenicol), allowing a broad range of
Agrobacterium or E. coli strains to be used. The pUC18
polylinker within the lacZa fragment allows blue/white screening of
clones in E. coli cloning work.

pCAMBIA1380 and 1390 are based on pCAMBIA1300, but the pUC18
polylinker-lacZa fragment has been deleted and replaced with the
simpler pUC8 (1380) and pUC9 (1390) polylinkers, which do not contain
potentially confounding start or stop codons. The full modular format is
provided for convenient PCR cloning and gene expression.

For researchers performing promoter analysis the use of the
minimal vector containing GUSPlus
(pCAMBIA
0305.2, which can be ordered through the BioForge
GUSPlus Project ) is recommended, rather than any of the other pCAMBIA
vectors. Co-transformation strategies are desirable to physically separate in
the transformed plant genome the promoter of the plant selection gene (usually
35S) and the promoter of interest (often much weaker or more specific) driving a
gus or other reporter gene. The way that we have been doing this for years is
that two vectors are separately transformed into the same strain of
Agrobacterium (or more preferably strains from the
BioForge TransBacter Project , for freedom to operate),
but obviously co-transformation can also be done by simultaneously transforming
with two separate isolates each containing one of the vectors. If using one
isolate, single colonies are selected, the plasmid DNA analysed, the cells
induced on special media (if required for your plant species), and the two cell
lines are mixed together immediately prior to application onto the plant tissues
to be transformed. In our hands this method gives 10-30% of transformed plant
lines containing both T-DNAs.

These vectors contain a fully functional gusA reporter construct for
simple and sensitive analysis of gene function or presence in regenerated plants
by GUS assay. The construct uses E.coli gusA (N358Q — to avoid
N-linked glycosylation) with an intron (from the castor bean catalase gene)
inside the coding sequence to ensure that expression of glucuronidase activity
is derived from eukaryotic cells, not from expression by residual
A.tumefaciens cells. The gusA reporter gene is cloned in new
modular format. These plasmids are suitable for insertion of other genes of
interest containing their own promoter and terminator. Researchers can excise
the gusA gene and insert their own gene of interest in its place or use
these vectors to create fusions of gusA with their gene of interest
(if you have created a pCAMBIA vector derivative that other researchers
will find useful and would like to share it, please let us know). These
vectors contain the pUC18 polylinker-lacZa and the same bacterial and
plant selection genes as their corresponding Minimal Selection Vectors.

pCAMBIA1302; pCAMBIA1303; pCAMBIA1304

For those desiring the best of both worlds in reporter genes we constructed
these vectors, similar in utility to the GUS Intron Selection Vectors (GIS), but
including GFP. Being a non-catalytic protein places an intrinsic limit on
detection sensitivity with fluorescent proteins and expensive equipment is
needed for quantitative assays and microscopic observation. GFP is nonetheless
popular as a reporter gene and we provide it cloned in full New Modular format
for those wishing to use it.

These vectors are based on pCAMBIA1301 (bacterial kanamycin resistance, plant
hygromycin selection, pUC18 polylinker in lacZa) but contain the
mgfp5 version of the Aequoria victoria green fluorescent
protein (Siemering et al., 1996) either alone - pCAMBIA1302 - or in
translational fusion with gusA (N358Q) in both arrangements:
pCAMBIA1303 has a gusA-mgfp5-His6 fusion, and pCAMBIA1304 has a
mgfp5-gusA-His6 fusion. These are intronless versions of gusA
(N358Q), so there is the possibility that expression in primary transformants is
the result of expression of the reporter proteins by residual Agrobacterium
tumefaciens cells or other bacteria present in plant cultures.

Analysis of large numbers of transformants of rice and Arabidopsis
at CAMBIA showed that the fluorescence produced by the MGFP5 protein was quite
faint. As a result of this our researchers constructed similar constructs using
the egfp gene available from Clontech. Results with these proteins were
far superior and, although we are unable to distribute vectors containing this
gene, we recommend that researchers purchase pEGFP from Clontech and use this to
construct their own plasmids analogous to pCAMBIA1302, pCAMBIA1303 or
pCAMBIA1304.

pCAMBIA1381 and 1391 and their Xa, b, c
ORF variants

Designed to utilize gusA as a true reporter of gene expression by
fusion construction, these vectors are derived from pCAMBIA1380 and 1390, and
contain a promoterless, non-intron gusA (N358Q) gene (without an
initiation codon) in three reading frames, and with either pUC8 or pUC9 oriented
polylinkers. This permits simple construction of carboxy-terminus protein
fusions to gusA. Plant selection is with hygromycin, and bacterial
selection with kanamycin.

The pCAMBIA1381 and 1391 vectors may also be used for construction of
transcriptional or translational fusions to gusA. They are similar to
the Xa, b, c series though they retain the initiation codon of the
NcoI site in the New Modular structure around the gusA (N358Q)
gene, and are only available in one reading frame.

pCAMBIA1281Z; pCAMBIA1291Z; pCAMBIA1381Z; pCAMBIA1391Z

Designed for promoter testing in planta, these vectors feature a
promoterless version of gusA (N358Q) with the catalase intron
immediately downstream of a truncated lacZa containing either the pUC8
or pUC9 polylinker. All plasmids in this series have hygromycin as the plant
selection gene, and bacterial selection is available with either chloramphenicol
(1281Z, 1291Z) or kanamycin (1381Z, 1391Z). The truncated lacZa is
functional for blue/white screening of clones in suitable E.coli host
strains.

Experience with these vectors has shown that the very strong 35S promoter (in
fact a double-enhancer version of it) which drives the hptII gene in
the T-DNA of these and most other pCAMBIAs causes significant interference in
the expression patterns observed. Interpret your results with
caution! Negative control transformants created using one of these
vectors without a promoter added upstream of the gusA gene show low to
moderate level GUS expression in a range of tissues. Enhancer-trap experiments
performed at CAMBIA using somewhat rearranged versions of these vectors have
also shown consistent interfering expression which we attribute to the nearby
35S promoter. This artefactual expression may be exacerbated in experiments
where researchers attempt to analyse trimmed-down versions of their promoters of
interest lacking the natural insulating sequences of full-length promoters.

To avoid this 35S-interference from promoter analyses, we recommend using
co-transformation strategies as described above in the section on the
Minimal Selection Vectors.

In such a strategy the promoter of interest can be cloned into one of our
Promoter Cloner Vectors, but this should first be modified by performing a
SmaI & XmnI double digestion, gel purification of the
large (~8.9kb) backbone fragment, and self-ligation. Such a vector might be
called pCAMBIA0381Z, but we have never constructed it for distribution as part
of the pCAMBIA vector kit.

Warning!! Agrobacterium use is constrained in jurisdictions such as
the USA, and it may be unwise to use it in any research that might result in a
product for sale or import into the USA. You may wish to use the Transbacter
system instead for freedom to operate. For information, see the BioForge
TransBacter Project.